Integrative Taxonomic, Ecological and Genotyping Study of Charophyte Populations from the Egyptian Western-Desert Oases and Sinai Peninsula

Present-day information available on the charophyte macroalgae in Egypt, including their phylogenetic affinities, remains largely incomplete. In this study, nine charophyte populations were collected from different aquatic biotopes across the Egyptian Western-Desert Oases and Sinai Peninsula. All populations were investigated using an integrative polyphasic approach including phylogenetic analyses inferred from the chloroplast-encoded gene (rbcL) and the internal transcribed spacer (ITS1) regions, in parallel with morphotaxonomic assignment, ultrastructure of the oospore walls, and autecology. The specimens identified belonged to the genera Chara, Nitella, and Tolypella, with predominance of the first genus to which five species were assigned though they presented some interesting aberrant taxonomic features: C. aspera, C. contraria, C. globata, C. tomentosa, and C. vulgaris. Based on our integrative study, the globally rare species C. globata was reported for the second time for the whole African continent. The genus Nitella was only represented by N. flagellifera, and based on the available literature, it is a new record for North Africa. Noteworthy, an interesting Tolypella sp., morphologically very similar to T. glomerata, was collected and characterized and finally designated with the working name ‘Tolypella sp. PBA–1704 from a desert, freshwater wetland’, mainly based on its concatenated rbcL+ITS1 phylogenetic position. This study not only improved our understanding on the diversity, biogeography and autecological preferences of charophytes in Egypt, but it also broadened our knowledge on this vulnerable algal group in North Africa, emphasizing the need of more in-depth research work in the future, particularly in the less–impacted desert habitats.


Introduction
Charophytes (Charales, Streptophyta), including both extant and fossil members of the order Charales (besides members of the extinct orders Sycidiales and Moellerinales), constitute an ancient group of terrestrial autotrophic macroalgae, the ancestors of which invaded land and developed to the present-day land plants 450 million years ago [1][2][3]. Ecologically, members of the family Characeae are widely distributed in freshwater and brackish biomes [4][5][6][7], with rare occurrence in marine habitats [8,9]. They play a keystone role in maintaining the balance and functioning of the ecosystems they colonize. Therefore, a better understanding of the ecological preferences of this vulnerable algal group is important for the conservation and restoration of their habitats [10,11]. Charophytes are known to be highly vulnerable to water pollution and eutrophication, and they therefore are one of

Phylogenetic Affinities of the Charophyte Specimens Investigated
To aid morphology-based identification process we assembled a dataset of 121 rbcL sequences of charophytes representing major genera (Table S1). The alignment included 70 Chara accessions, 32 Nitella sequences, 15 Tolypella sequences, and 4 Lamprothamnium species. Representatives of these genera formed robust (Tolypella) or strongly supported (Nitella and Lamprothamnium) generic clades. Chara was resolved only topologically as a sister of Lamprothamnium (98/1.00; Figure 1). All our Chara sequences were assigned to well-supported species clades (C. vulgaris, C. globata, C. contraria, C. aspera, and C. tomentosa). Similarly, in the genus Nitella the new sequence was placed in the robust N. flagellifera clade. Only our Tolypella accession occupied unresolved position in a weakly supported clade. In the analyses with the concatenated data set that included 16 rbcL and ITS1 sequences of Tolypella, our sequence showed weak affinity to Tolypella sp. from Australia ( Figure 2). Combined chloroplast and nuclear markers provided additional support for many internal clades in the genera Nitella ( Figure 3) and Chara (Figure 4), and also confirmed affinities of N. flagellifera, C. aspera, and C. contraria.

Morphotaxonomy, Autecology, and Biogeography of the Charophyte Specimens Studied
In the present study, seven taxa belonging to the genera Chara (C. aspera, C. contraria, C. globata, C. tomentosa, and C. vulgaris), Nitella (N. flagellifera), and Tolypella (Tolypella sp. PBA-1704) were identified and discussed from the standpoints of morphotaxonomy and ecological characterization. The worldwide rare species C. globata is herein reported for the second time in the whole African continent. Interestingly, N. flagellifera represents the first record for both Egypt and North Africa. An interesting Tolypella sp., morphologically similar to T. glomerata, is designated with the working name 'Tolypella sp. PBA-1704 from a desert, freshwater wetland', mainly based on its concatenated rbcL+ITS1 phylogenetic position. Detailed descriptions, ecological preferences, and biogeography of all these taxa are given in the following. Hydrochemical characteristics of the habitats studied are provided in Table 1.
• Remarks: Our specimens are consistent with the diagnosis of the protologue illustrated in Wood and Imahori [47,53]. Besides the clear-cut differences in some morphotaxonomic features with the most morphologically close species C. vulgaris (in particular tylacanthous cortication in C. contraria vs. aulacanthous in C. vulgaris), the two species are also well separated genetically ( Figure 1).    • Description: Plants green to olive green ( Figure 8A,B), monoecious, 20-85 (-95) cm tall, unencrusted to heavily incrusted, forming a massive growth inside the main springhead and the outlet channel of thermal mineral desert spring ( Figure S3A,B). Axes predominately stout, 610-1580 (-2000) µm in diameter ( Figure 8C and Figure S3C). The internodes usually longer than the branchlets, 1.5-4 (-5) times longer than the branchlets, up to 8 cm long ( Figure 8A-C and Figure S3C), the upper parts of thalli look like spherical loose heads ( Figure 8C,D). Cortex irregularly diplo-to triplostichous, slightly isostichous to distinctly tylacanthous ( Figure 8F-I and Figure S3F). Spine-cells mostly solitary ( Figure 8F,H and Figure S3E) or rarely in a bunch of four (only one very long and the other surrounding three distinctly very short) ( Figure 8G), subulate, with thickened cell walls at their ends. Stipulodes diplostephanous (in 2 tiers), 2 sets per branchlet, well developed, long aculeiform with acute ends ( Figure 8E and Figure S3D). The branchlets usually straight, but still slightly arcuate, 9-10 in a whorl, 1.5-2 (-2. General distribution and ecology: Rare, but still flagship, temperate species with disjunctive biogeographical distribution, particularly in the arid and semiarid regions. Fresh-brackish, moderately alkaliphilic (pH: 7.1-8.0) species preferring waters rich in sodium sulphates, and calcium/magnesium bicarbonates [43]. So far, it has only been recorded in Asia (China, Iran, Israel, Kazakhstan, Kyrgyzstan, Russia, Turkmenistan and Uzbekistan) [43,47,[54][55][56][57], Europe (Romania and the European part of Russia) [57,58], the Sahara-Arabian Desert in Sinai Peninsula [43], and in North Africa (only in Tunisia) [57]. During the present study, C. globata was found in the thermal mineral desert spring 'Ain Wazedi' in the Siwa Oasis. This Saharan biotope was characterized by the following hydrochemical characteristics:-high water temperature ( The diagnostic taxonomic features of the Siwa C. globata population fitted better the specimens recently described by Romanov et al. [43] than the protologue redescribed by Wood and Imahori [47,53]. However, our specimens still differ from the description in Romanov et al. [43] by the following taxonomic features: (1) stem cortex irregularly diplo-to triplostichous, slightly isostichous to distinctly tylacant-hous (vs. consistently a tylacanthous diplostichous stem cortex), (2) spine-cells mostly solitary, long acuminate and rarely in a bunch of four (only one very long and the other surrounding three distinctly very short) (vs. only solitary and variable in length from short conical-papillose to conical to long subulate), (3) gametangia usually present at the 3 lowest nodes of the corticated segments (vs. gametangia occurring at the 2-4 lowest nodes between corticated segments and rarely between ecorticate segments), and (4) ripe oospores are obviously dominant (vs. oospores low or absent in the majority of the specimens). We think that all these phenotypic variations are environmentallyinduced and with a low taxonomic value. Taxonomically, Romanov et al. [43] also proposed that C. globata should be transferred and assigned to the subsection Chara in the section Chara, instead of the section Grovesia having a triplostichous stem cortex, in terms of the taxonomic observations obtained (i.e., consistently and generally tylacanthous diplostichous stem cortex, solitary spine-cells, and stipulodes in two tiers), corresponding well to the section Chara [47]. On the contrary, the Siwa C. globata specimens investigated in the present study are mainly characterized by the presence of isostichous to tylacanthous diplo-to triplostichous stem cortex ( Figure 8F-I).
Ling et al. [55] also documented irregular triplostichous tylacanthous cortex in Chinese specimens of C. globata. Additionally, the subsection Chara placement proposed by Romanov et al. [43] was not supported by crossing experiments conducted by Proctor [59,60], who pointed to the affinity of C. globata towards the subsection Hartmania. However, the combined morphotaxonomic and phylogenetic data obtained in this study (Figure 1), as well as work of Romanov et al. [43], showed that C. globata has more or less a closer affinity to species of the subsection Hartmania but that it is still different genetically and taxonomically (in particular in the presence of the verticillate bract-cells and arcuate branchlets mainly in the apical parts of thalli). In our opinion, the accurate taxonomic placement of C. globata is still problematic and more integrative studies are needed.    Our observations on the oospore wall ornamentation (mainly smooth to pustular, to slightly papillate, evenly covering fossa and ridges) coincide with the findings of Romanov et al. [43], and confirm one of the key diagnostic features for this rarely investigated species. In spite of C. globata having been recently recorded for the first time in North Africa in Tunisia, its morphotaxonomic diagnostic traits were poorly revealed (Figures 1c  and 2e,f in [57]). In our polyphasic study, we are providing detailed information on the morphotaxonomy and on the phylogenetic affinity of the C. globata population in the Siwa Oasis, and these observations are novel for the whole African continent. Based on the rbcL phylogenetic analysis, C. globata is genetically distinct from the morphologically most allied taxa in the subsection Hartmania, such as C. polyacantha, C. hispida, C. rudis, C. baltica, C. intermedia, and C. horrida, and also placed separately within a clade that included only representatives of this geographically-limited species from Egypt and Israel (Figure 1). From the ecological standpoint, the Siwa Oasis C. globata population was found in the thermal mineral spring 'Ain Wazedi' and it can be considered as a flagship species in this unique biotope. This observation coincides with the findings of Romanov [57], who as well recorded this charophyte in an oasis-like locality in Tunisia. Spring habitats are well established as biodiversity hotspots, often also hosting rare and highly-specialized algal species [34,61]. It should also be recalled that Romanov et al. [43] recorded C. globata in the Sinai Peninsula, and emphasized rather little knowledge on the diversity of charophytes in the Sahara-Arabian Desert, indicating it as worthy of further studies. C. globata seems to be highly adapted and widely distributed in the Egyptian desert habitats (A.A.S. and co-workers, unpublished data), and it could therefore be considered as one of the characteristic Chara populations not only for Egypt but also for North Africa and the Sahara-Arabian Desert in general. We also think that the only available records of C. hispida var. hispida f. polyacantha (A.Braun) R.D.Wood and C. hispida var. baltica (Bruzelius) R.D.Wood from the Siwa Oasis [36] are misidentifications and indeed belong to C. globata. Although the morphotaxonomic traits of both taxa are not available in Corillion and Guerlesquin [36] for an in-depth check, they were sampled from the same oasis and share some morphological taxonomic features with C. globata. We predict that C. globata might be recorded in the future in the other Maghreb countries, particularly by applying combined morphological and phylogenetic approaches. Accordingly, it has been established that subtle species identification of members of the charophytes at the species and intraspecific level has nowadays become much easier thanks to the integrative polyphasic approaches, irrespective of the occurrence of populations showing marked phenotypic variability and developing so-called "phenoecodemes" as a result of the environmental and/or culture conditions [22,29,44].
Heads not formed. Gametangia conjoined at the 1st-3rd lowest branchlet nodes, without mucous ( Figure 12C Figure 12M). • Distribution in Egypt: This is the first record of this charophyte both in North Africa and in Egypt.

•
Remarks: This species is considered a new record for Egypt and also for North Africa based on the published literature ( [5,32,36,47] and references therein). Our N. flagellifera rbcL and ITS1 gene sequences are also the first ones for North Africa. From the taxonomic and phylogenetic points of view, our N. flagellifera specimens coincide with the specimens redescribed by Wood and Imahori [47] and also with the findings of Borges and Necchi [25]. Noteworthy, gametangia in our study were noticed at the first node of the branchlet ( Figure 12C), and this taxonomic observation has also been documented for the Brazilian N. flagellifera population investigated by Borges and Necchi [25], and other previous studies (e.g., [66,67]). Contrarily, Wood and Imahori [47] noted the lack of gametangia at this position. Blindow et al. [69] pointed out the presence of high phenotypic plasticity and some taxonomic discrepancies in the key characters of the Subfamily Nitelleae, which hamper species identification. N. flagellifera also resembles morphologically and phylogenetically the closest species N. oligospira [25,47]. However, N. flagellifera is still different taxonomically by having a secondary central ray ( Figure 12C,D), a unique taxonomic feature that can be easily used to distinguish it from N. oligospira. These two species have more or less similar distribution patterns, are phylogenetically closely related, and also occupy a distinctive position in the genus tree ( Figure 3).  • Description: Plants monoecious, pale green to green, unencrusted, fragile, up to 22 cm tall, with few coarse heads. Axes moderately slender, 500-850 µm in diameter. Internodes 1-2 times as long as the branchlets, becoming shorter towards the apex, up to 5 cm long. Sterile and fertile branchlets different ( Figure 13A-D). The first node of the main axis produces 6-7 sterile branchlets and 2-4 secondary axes. The sterile branchlets are undivided and in a series of 3-5 elongated cells ( Figure 13B,C). The fertile whorls produced by the secondary axes, short and grouped into fertile heads. Heads few to numerous, 3-14 per shoot ( Figure 13A-D). The fertile branchlets apparently consist in a central row of cells (the "rachis") that is a succession of nodes and internodes. These nodes carry the gametangia as well as 3 rays of 2-3 cells. All terminal cells are elongated, obtuse ( Figure 13E). Gametangia conjoined at the fertile branchlet nodes, usually 1 central adaxial antheridium with 1-2(-3) lateral oogonia ( Figure 13F). Oogonia 275-335 µm (incl. coronula) long × 250-280 µm wide, with 8-9 convolutions; coronula 30-35 µm high × 40-55 µm wide. Oospores brown to golden brown to slightly dark brown, (275-)320-354 µm long × 215-241 µm wide ( Figure 13G); striae of 7-8 prominent, flanged ridges ( Figure 13H,I); fossae and ridges with smooth ornamentation ( Figure 13J); fossae 37-43 µm across. Antheridia solitary, small, sessile, 105-140 µm in diameter ( Figure 13F). • Distribution in Egypt: This is the first record worldwide of this genetically distinctive charophyte. We therefore designated it with the working name 'Tolypella sp. PBA-1704 from a desert, freshwater wetland' mainly based on its concatenated rbcL+ITS1 phylogenetic placement.  (Table 1). • Remarks: In spite of the high morphotaxonomic similarities between our Tolypella specimens and the cosmopolitan species T. glomerata [47], it is apparently still distinct phylogenetically from that taxon (Figures 1 and 2), and we therefore designated it with the working name 'Tolypella sp. PBA-1704 from a desert, freshwater wetland' mainly based on its concatenated rbcL+ITS1 phylogenetic placement. Further in-depth taxonomic and molecular studies on this interesting Tolypella taxon are necessary to propose it as a (morphologically) cryptic species new to science or to recognize it as belonging to a wide genetic variability of T. glomerata.

Charophyte Sampling, Processing, and Morphological Identification
During sampling campaigns conducted from October 2016 to May 2018 to unravel the hidden phycological diversity in the Egyptian Oases and other comparable habitats, the charophyte populations investigated in the present study were collected from different aquatic biotopes, including thermal mineral desert springs, agricultural ditches, and shallow wetlands in the Western Desert Oases (Siwa, El-Dakhla, and El-Farafra) and a nutrient-rich artificial muddy pool in the mountain valley "Wadi El-Arbaeen" in the Sinai Peninsula, Egypt (Table 2; Figures S1 and S2). The major water source in the Western Desert Oases is the Nubian Sandstone Aquifer System (NSAS), the world's largest fossil freshwater reservoir [70]. In the Sinai Peninsula, only one population of Chara contraria could be sampled from Wadi El-Arbaeen. It is a mountain wadi located in the UNESCO world heritage site "Saint Catherine Protectorate". The only water source in this mountain valley is the shallow aquifers that are typically recharged by heavy rainfalls [71]. Charophyte specimens were collected in clean sterile polyethylene terephthalate bottles and then transported to the laboratory where the specimens were cleaned with tap water to be carefully analyzed under the light microscope. A part of each specimen collected was also dried for the DNA extraction and sequencing. The specimens were identified following primarily Wood and Imahori [47,53] and Krause [4]. The key morphotaxonomic characters were checked and determined with the aid of a Novex ® RZT stereomicroscope (EUROMEX microscopes BV, Arnheim, the Netherlands), and a BEL ® photonics biological light microscope (BEL ® Engineering, Monza, Italy), and the light microscopy (LM) micrographs were taken with a Canon Powershot G12 digital camera. The biometric data provided were based on a minimum of 20-25 measurements for each character per species. The oospores were treated with acetic acid to remove any lime-shell, washed with distilled water and cleaned from the spiral cells by adding 10% Triton X100, and then stored at 60 • C for at least 10 h [43]. They were washed again with distilled water and sonicated to completely get rid of the spiral cells. The cleaned oospores were stored in 95% alcohol. To characterize detailed architecture of the oospore walls they were mounted, air-dried onto small round aluminum stubs, sputtered with chromium (Cr), and then studied with a Sigma ® 300 VP electron microscope (Carl Zeiss AG, Oberkochen, Germany) at 3.0-20.23 kV at the A.V. Zhirmunsky National Scientific Center of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences, Vladivostok, Russia. The terminology used to describe the oospore surface follows Urbaniak [72]. All photos were digitally manipulated, and plates were created using Adobe

DNA Extraction, Amplification, and Sequencing
Total genomic DNA was extracted as described by Echt et al. [75] with some modifications [76]. Polymerase chain reaction (PCR) amplification was performed using the Encyclo Plus PCR kit (Evrogen, Moscow, Russia) with a T100 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). The rbcL gene was amplified and sequenced in two fragments, using the following primer pairs for PCR: rbcL-RH1 [77] and rbcL-972R, for the 5 -gene fragment; and rbcL-295F [78] and rbcL-1379R ( [79] with modifications) for the 3 -fragment. The PCR cycling profile included an initial step of 3 min at 95 • C, followed by 38 cycles of denaturation at 95 • C for 20 s, 20 s of annealing at 49 • C, and 1 min at 72 • C, with a final extension at 72 • C for 5 min. The ITS1 rDNA region was amplified using primers ITS-36F and ITS-IR [80]. The PCR cycling profile for this region included a denaturation at 95 • C for 3 min, followed by 38 cycles of denaturation at 95 • C for 20 s, annealing at 55 • C for 20 s, elongation at 72 • C for 1 min and a final extension step at 72 • C for 5 min. The PCR products were purified by ExoSAP-IT PCR Product Cleanup Reagent (Affymetrix, Santa Clara, CA, USA) and sequenced in both directions using an ABI 3500 genetic analyzer (Applied Biosystems, Foster City, CA, USA) with a BigDye terminator v3.1 sequencing kit (Life Technologies Corporation, Austin, TX, USA) and the same primers used for PCR. Sequences were assembled with the Staden Package v1.4 [81], aligned manually in the SeaView program [82]. The sequences were deposited in GenBank (Table 2).

Phylogenetics Analyses
Maximum likelihood (ML) analysis was carried out using PAUP 4.0b10 [83]. Bayesian inference (BI) was performed using MrBayes 3.1.2 [84]. To determine the most appropriate DNA substitution model for our datasets, the Akaike information criterion (AIC; [85]) was applied with jModelTest 2.1.1 (Table 3; [86]). ML analysis was done using heuristic searches with a branch-swapping algorithm (tree bisection-reconnection). Some parameters of ML and BI were listed in Table 3. In BI, convergence of the two chains was assessed, and stationarity was determined according to the 'sump' plot with the first 25% of samples discarded as burn-in; posterior probabilities were calculated from trees sampled during stationary phase.

Conclusions
This study improved and updated our understanding on the taxonomic status, species diversity and autecological niches of nine charophyte populations colonizing different biotopes in the Egyptian Western-Desert Oases (North Africa) and Sinai Peninsula. Nitella flagellifera is here recorded for the first time in Egypt and North Africa.
An interesting Tolypella sp. has been designated with the working name 'Tolypella sp. PBA-1704 from a desert, freshwater wetland', based on its distinct position in rbcL+ITS1 placement from the morphologically similar T. glomerata. In spite of the fact that most Chara taxa we recorded are cosmopolitan and eurytopic [4,47], our integrative study confirmed the occurrence of the worldwide rare species C. globata for the second time in North Africa. The surveys carried out in the present study have also made it possible to provide further information for some species already reported from Egypt, such as C. aspera, C. contraria, C. tomentosa, and C. vulgaris [32,36,44]. It should be stressed that most of the localities from which the aforementioned taxa had been previously reported from Egypt during the 6th and 7th decades of the last century have nowadays been degraded and often disappeared as a result of the immense human-mediated pressures and of the lack of governmental legislation to conserve this severely threatened algal group. In agreement with our conclusion, the recent study by Mjelde et al. [11] on the charophytes in Myanmar highlighted that eutrophication and direct human pressures on the freshwater habitats are among the main factors reducing charophyte diversity. Blindow [12] pointed out that eutrophication can cause competition among submerged macrophytes, a case which is physiologically unfavorable to the vulnerable species of charophytes.
In accordance with the SRP-based trophic system proposed by Lambert-Servien et al. [14] for the charophytes, the charophyte habitats we studied can be classified as meso-eutrophic, and, rarely, hyper-eutrophic (see TP and SRP values in Table 1). Therefore, the charophyte species identified can be considered eurytopic and P-enrichment-tolerant species. The relationships between charophyte distributional patterns and environmental variables, in particular nutrients, have been discussed in several previous studies [15,16,89]. To broaden our knowledge on this vulnerable group of algae in Egypt and North Africa, further studies applying polyphasic approaches based on sampling campaigns, in particular from the remote and isolated desert environments and from moderately impacted urban habitats, are needed. Ultimately, characterizing the eco-physiological adaptive strategies of this streptophycean group of algae is of pivotal importance to fill knowledge gaps about the mechanisms of their acclimatization to their harsh environmental conditions. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/plants10061157/s1. Figure S1: General views on sampling sites from which the different charophyte populations were collected: (A-C) Chara aspera population in a mineral spring-fed agricultural ditch in the Siwa Oasis; (D-F) C. contraria population found in a nutrient-rich pool in the mountain valley "Wadi El-Arbaeen", Saint Catherine Protectorate, South Sinai; (G,H) an agricultural ditch in the El-Dakhla Oasis where the C. contraria population was sampled; (I-L) the thermal mineral desert spring 'Ain Wazedi' in the Siwa Oasis where the biogeographically limited species C. globata was sampled. This relatively stiff and heavily encrusted population formed a massive growth inside the springhead and its outlet channel. Figure Table S1: Datasets of rbcL and ITS1 sequences of charophytes included in the present study. Sequence-alignment-data (4 FASTA files in zipped directory).